Detection and quantification of N6-methyladenosine in messenger RNA by TLC

Abstract

The base-modified nucleotide, N6-methyladenosine, is a relatively abundant modification found in the mRNA of most higher eukaryotes. Methylation levels can change dependent upon environmental conditions, cell differentiation state, or following knockdown of members of the methylase complex, and it is often useful to directly measure and compare N6-methyladenosine levels between samples. Two dimensional chromatography of radiolabeled nucleotides, following specific nuclease treatments, provides a robust, sensitive, and reproducible assay for this modification.